Methods controlling gene expression

Abstract

The present invention is in the field of genetics, especially plant genetics, and provides agents capable of controlling gene expression. The present invention specifically provides sequences of naturally occurring, tissue-specifically expressed microRNAs. The invention further provides for transgenic expression constructs comprising sequences encoding said microRNAs. By incorporation of the microRNA encoding sequence the expression from said expression construct is specifically silenced in the tissue where the naturally occurring microRNA is naturally expressed. Thereby the expression profile resulting from the promoter is modulated and leakiness is reduced. The invention further provides for a method for modulating transgenic expression by incorporating sequences encoding said microRNAs into transgenic expression constructs. The compositions and methods of the invention can be used to enhance performance of agricultural relevant crops and for therapy, prophylaxis, research and diagnostics in diseases and disorders, which afflict mammalian species.

The invention claimed is: 
     
       1. A method for suppressing or lowering expression of a chimeric nucleotide sequence in a plant, comprising the steps of:
 a) providing an expression construct comprising a promoter sequence functional in a monocotyledonous plant and functionally linked thereto a chimeric nucleotide sequence to be expressed, wherein said chimeric nucleotide sequence comprises:
 i) at least one sequence comprising an open reading frame encoding a protein that is capable of conferring an agronomically valuable trait in a plant, and 
 ii) at least one sequence substantially complementary to a microRNA sequence naturally occurring in said plant, 
 wherein the at least one sequence of i) and the at least one sequence of ii) are heterologous to each other, 
 
 b) introducing said expression construct into a plant, and 
 c) expressing said chimeric nucleotide sequence in said plant, 
 wherein the plant is a monocotyledonous crop plant, wherein the expression of said chimeric nucleotide sequence is suppressed or lowered in tissues where said naturally occurring microRNA is expressed, and wherein said chimeric nucleotide sequence expression is not suppressed or lowered in tissues where said naturally occurring microRNA is not expressed. 
 
     
     
       2. The method of  claim 1 , wherein said promoter is selected from the group consisting of constitutive promoters, tissue-preferential promoters, developmental promoters, and inducible promoters. 
     
     
       3. The method of  claim 1 , wherein the at least one sequence being substantially complementary to the microRNA has an identity of at least 60% or not more than 6 mismatches over its entire sequence in comparison to the complement of the microRNA sequence. 
     
     
       4. The method of  claim 3 , wherein said mismatches are in the region corresponding to the 3′-region of said microRNA sequence. 
     
     
       5. The method of  claim 1 , wherein the microRNA is tissue-specific expressed, spatially-regulated, developmental regulated, and/or regulated by biotic or abiotic stress factors. 
     
     
       6. The method of  claim 1 , wherein said expression construct is in a plasmid. 
     
     
       7. The method of  claim 1 , wherein the microRNA has a natural expression profile in the plant selected from the group consisting of
 a) expression in any tissue but no expression in seed, 
 b) expression in seeds but not in other tissues, 
 c) drought or other abiotic stress-induced expression, 
 d) plant pathogen-induced expression, and 
 e) chemical induced expression. 
 
     
     
       8. The method of  claim 1 , wherein the microRNA is a plant microRNA selected from the group consisting of
 a) the sequence as described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, or 266, and 
 b) derivatives of the sequence described in a) having at least 70% identity to the sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, or 266. 
 
     
     
       9. The method of  claim 1 , wherein the at least one sequence being substantially complementary to the microRNA is positioned in a location of the nucleotide sequence to be expressed corresponding to the 5′-untranslated region or the 3′-untranslated region of said nucleotide sequence. 
     
     
       10. The method of  claim 1 , wherein the agronomically valuable trait is selected from the group consisting of disease resistance, pest resistance, herbicide resistance, sterility, grain characteristics, vigor, time to harvest, enhanced nutrient content, novel growth patterns, flavors or colors, and salt, heat, drought, and/or cold tolerance. 
     
     
       11. The method of  claim 1 , wherein the at least one sequence capable to confer an agronomically valuable trait in a plant is selected from the group consisting of genes involved in the synthesis and/or degradation of proteins, peptides, fatty acids, lipids, waxes, oils, starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids, hormones, polymers, flavinoids, storage proteins, phenolic acids, alkaloids, lignins, tannins, celluloses, glycoproteins, and glycolipids.